Spiritual Holy Water Sites in Ethiopia: Unrecognized High-Risk Settings for Transmission of Pulmonary Tuberculosis

Ethiopia is a high-tuberculosis (TB) burden country with 157 new cases per 100,000 people, with 23,800 TB-related deaths in 2020. In Ethiopia, TB patients have different healthcare-seeking behaviors. They frequently visit spiritual places, such as holy water sites (HWSs), to seek treatment for their illness spiritually. This study examined the prevalence of pulmonary TB (PTB) and drug susceptibility profiles of Mycobacterium tuberculosis (MTB) isolates among spiritual HWS attendees in Northwest Ethiopia. A cross-sectional study was conducted from June 2019 to March 2020. Sputum samples were collected, processed, and cultured using Löwenstein–Jensen (LJ) culture medium. Second-generation line probe assays (LPAs), GenoType®MTBDRplus VER2.0 and GenoType®MTBDRsl VER2.0, were used to detect anti-TB drug-resistant isolates. STATA 17 was utilized to perform descriptive statistics, bivariate, and multivariate regression analyses. Of 560 PTB-symptomatic participants, 21.8% ((95% confidence interval (95 CI): 18.4–25.2%)) were culture-positive, resulting in a point prevalence of 1,183/100,000 attendees. Amongst HWS attendees, culture-positive TB occurred most commonly in persons 18–33 years of age (28.5% (95 CI 23.4–34.3%)). Other participant characteristics significantly associated with culture-positive PTB were as follows: rural residents (adjusted odds ratio (aOR) 2.65; 95 CI 1.38–5.10), married participants (aOR 2.43; 95 CI 1.28–4.63), family members >5 per household (aOR 1.84; 95 CI 1.04–3.24), and sharing living space (aOR 10.57; 95 CI 3.60–31.13). Also, among 438 participants followed for 12 months after showing negative TB culture results while at the HWS, 6.8% (95 CI 4.4–9.4%) developed or contracted culture-positive TB post-residency at the HWSs. Of the 122 tested isolates, 20 (16.4%) were isoniazid (INH) and/or rifampicin (RIF) resistant. Multidrug-resistant (MDR) TB was detected in 15 cases (12.3%), five of which were fluoroquinolones (FLQs) resistant. The findings from this study should raise a concern about HWSs as potential high-risk settings for TB transmission. It is recommended that appropriate control measures be instituted that include compulsory TB testing and tightened infection control at HWSs, where an increased risk exists for transmission of TB.


Introduction
Tuberculosis (TB) remains to be a major global health issue [1,2].Worldwide, "10.0 million people were infected with TB and over 1.5 million died" from it in 2020 [2].Until the 2020 coronavirus disease (COVID-19) pandemic, TB was the major infectious agent-related cause of mortality globally [2].Although TB is a global issue, its prevalence, public health, and economic impact vary greatly between nations [1,2].TB is the leading cause of mortality among infectious diseases in low-and middle-income countries (LMICs) due to several factors.Poor healthcare access, overcrowded living conditions, poor nutrition, and the HIV/AIDS pandemic all contribute to the high burden of TB in LMICs [2][3][4].Ethiopia, like other LMICs, has a high burden of TB, which poses challenges to its public healthcare system [2,5,6].Ethiopia's TB incidence was 157 per 100,000 persons, with 23,800 people dying from the disease in 2020 [1], suggesting that TB continues a signifcant cause of death in the country.
Te emergence of drug-resistant TB (DR-TB) has posed a signifcant threat to global and national TB control eforts, as DR-TB has the potential to spread globally, emphasizing the need for additional prevention and care measures [1,2].A recent national report revealed that the prevalence of MDR or RIF-resistant TB (MDR/RR-TB) in Ethiopia was 0.71% among newly diagnosed TB cases and 12.0% in retreated TB patients [1].Besides, "pre-extensively drugresistant (pre-XDR)" TB and "extensively drug-resistant (XDR)" TB were 5.7% and 0.6%, respectively [7].Since the country lacks the facility to undertake universal drug susceptibility testing (DST) on all incident TB cases, early TB case detection and initiation of proper treatment are problematic in many public health facilities in Ethiopia [8].
Early TB case detection, successful TB patient treatment according to international standards, and TB prevention are global plans to halt TB and reduce deaths and transmission [2,9].However, "a key obstacle to achieving this goal has been that many people with TB are currently being" missed by healthcare systems [1].Globally, over 2.9 million TB cases were anticipated to be underdiagnosed or diagnosed but unreported, most of which occurred in LMICs with weak healthcare systems [1].Finding these cases, efectively diagnosing them, and commencing appropriate treatment are critical to containing the disease [1,10].In Ethiopia, about two-thirds of individuals with active TB in the community remain undiagnosed and thus untreated [11,12].Tis could be because the Ethiopian TB prevention and control program relies mostly on passive case fnding, which requires TB suspects to self-present and visit public healthcare facilities [12].However, studies showed that individuals with TB symptoms in LMICs, such as Ethiopia, exhibit diferent healthcare-seeking behaviors and use other methods before seeking treatment at public healthcare facilities [13][14][15].Besides, people from poor communities have insufcient knowledge about TB disease, and they often confuse the symptoms of TB as indicative of other infections [14].Moreover, healthcare facilities are not nearby to give TB services, and people with TB symptoms often live in places where government services have a hard time reaching, due to travel costs and fear of stigma, TB patients may not visit public healthcare facilities for diagnosis and treatment [14,15].Tus, TB patients can delay the opportunity for early diagnosis and the initiation of appropriate treatments.Tis can intensify and threaten TB transmission in households and the community.
In Ethiopia, people use spiritual holy water as an alternative treatment for a variety of diseases, including respiratory ailments [13,[16][17][18].Holy water sites (HWSs) are designated areas with springs that are believed to have the power to cure various types of illness [13].Although the number of people who use holy water as a treatment option is not well documented in the country, it is known that attendees believe in its healing power and use it as an alternative treatment for various illnesses.Particularly, Ethiopian Orthodox Tewahedo Christians have unwavering faith in the curative power of spiritual holy water [13,16].Tus, individuals from diferent parts of the country travel to HWSs to seek the curative power of holy water blessed by Orthodox priests and they reside in shared living spaces (rooms) for a certain period.Te rooms are built as temporary waiting spaces, and they are small, overcrowded, and not well-ventilated, which can increase the risk of TB and other respiratory disease transmissions [13,19].
In the Amhara region, the focus of this study, over 82.5% of the population are Ethiopian Orthodox Tewahedo Christian followers [13,20], and faith-based therapy with spiritual holy water for TB and other diseases is widely practiced [13].Although studies show that TB patients seek care from traditional healers and spiritual HWSs, the burden of TB and the drug resistance pattern of MTB isolates among HWS attendees in Ethiopia have not been thoroughly investigated.To the best of our knowledge, one earlier study has shown that "the prevalence of PTB among HWS attendees was 7.4-fold higher than the prevalence in the general population in Ethiopia" [13].However, this study covered a limited geographical area and used smear microscopy to diagnose TB in symptomatic persons; given the low sensitivity of this technique, its fndings might not refect the true TB burden among this cohort of populations in the region.Hence, identifying such special congregate settings in the indigenous communities, considering these settings as hotspot sites for TB transmission, and conducting a systematic TB screening would aid in reaching those unreachable, missed, or undiagnosed TB cases for timely diagnosis and care.Terefore, this study aimed to assess the prevalence of TB and drug susceptibility profles of MTB isolates among individuals with symptoms of PTB attending spiritual HWSs in the Amhara region, Ethiopia.

Study Setting.
Te Amhara region is located in the northwestern parts of Ethiopia and comprises eleven zones and three administrative towns (Figure 1).A cross-sectional study was done between June 2019 and March 2020 in nine purposefully selected HWSs found across nine administrative zones in the region.One HWS was chosen from each of the study zones.Te HWS in each zone was chosen based 2 International Journal of Microbiology on its consistent popularity for holy water treatment, its ability to accommodate many attendees, and where many people visit it throughout the year and stay for an extended time [13,18] (Figure 1 and Table S1).

Population, Study Participants, and Recruitment.
Te study population comprised all attendees at the HWSs during the data collection period [18].Following TB screening criteria, individuals with a persistent cough lasting two weeks or longer and other PTB-suggestive symptoms, such as productive cough or expectorating blood-containing sputum, fever, chest pain, shortness of breath, fatigue, night sweating, loss of appetite, unexplained weight loss, and contact history with active TB patients and history of TB disease, were screened [5].A total of 10,313 attendees (≥18 years of age) were screened at nine selected HWSs during the study period; 560 of these individuals exhibited symptoms of PTB and participated.Te study settings, total attendees screened for PTB-suggestive symptoms, anticipated PTB-symptomatic attendees from each study site, total participants, and laboratory test results are depicted in the additional fles (Table S1).

Eligibility Criteria.
Inclusion criteria: Individuals who were ≥18 years of age and fulflled the screening criteria were included in the study [5,21].Exclusion criteria: Individuals who were seriously ill and unable to provide sputum samples and other relevant clinical or demographic information were excluded.Moreover, individuals who were receiving anti-TB treatment during data collection, and those whose permanent address was outside of the study region were excluded.

Screening and Socio-Demographic Data Collection.
All attendees were screened for symptoms suggestive of PTB, following the guidelines [5,21].Trained nurses and medical laboratory technologists with experience in TB screening and similar feld data collection perform the data collection.Descriptive demographic data were collected from eligible HWS attendees by interviewer-administered questionnaire.
Participants' socio-demographic data included their sex, age, marital status, educational status, household size, and place of residence, as well as risk factors for PTB infection such as a history of TB disease, contact with active TB patients or person with a chronic cough, family history of TB disease, previous HWS visits (the last one year), the number of days International Journal of Microbiology spent at the HWS, and shared living space (room) at the HWSs, were recorded.

Culture and Identifcation
Test.Sputum specimens from attendees with PTB symptoms were obtained using sterile and leak-proof collection containers [18].Te sputum specimen was placed in an icebox and delivered to the Amhara Public Health Institute, a regional public health referral laboratory center, Bahir Dar, Ethiopia [18].Sputum specimens were prepared and inoculated into a Löwenstein-Jensen (LJ) culture medium following standard laboratory procedures.Ziehl-Neelsen (ZN) staining was done to confrm all LJ culture-positive isolates [22].Te MPT64 antigen test (Capilia TB-Neo, TAUNS Laboratories, Inc., Japan) was used to diferentiate MTB complex species from non-TB mycobacteria (NTM) [23].
2.6.Specimen Preparation.After subculturing, suspensions were prepared from LJ-positive specimens and transferred into 1.5 ml of PrimeStore Molecular Transport Medium (PS-MTM; Longhorn Vaccine and Diagnostics, San Antonio, Texas, USA).Preparing suspensions of MTB colonies from LJ culture media depended on the culture state [24].In brief, for intact slopes, colonies were gently scraped of using an inoculation loop and suspended (washed down) in 1 ml of sterile water in the original culture bottle.After pipetting of the suspension, it was transferred into a 1.5-ml Eppendorf tube and then transferred to the 1.5 ml PS-MTM [24].Mycobacterial suspensions prepared in PS-MTMP tubes were transported to South Africa by air at ambient temperature for other genotyping procedures.

DNA Extraction.
Te MTB DNA was extracted from all 122 LJ-positive isolates using the PrimeXtract ™ kit (Long- horn Vaccines and Diagnostics, San Antonio, TX, USA) following the manufacturer's instructions [25].Briefy, 200 μL of 100% ethanol, 200 μL of lysis bufer, and 200 μL of MTB inoculum (preserved in PS-MTM) were transferred into a 1.5-mL microcentrifuge tube, then vortexed and centrifuged.Te entire supernatant was transferred to a microextraction column and centrifuged at 13,000 rpm for 1 minute, and the fow-through material was discarded.Wash bufer 1 (500 μL) was applied to the extraction column and centrifuged at 13,000 rpm for 1 minute, followed by further addition of wash bufer 2 (500 μL) to the extraction column and subsequent centrifuging as described above, discarding the fow-through material.Ten, DNA was eluted by 1 minute of centrifugation at 13,000 rpm using 50 μL of preheated (60-70 °C) elution solution [25].For future use, the extracted MTB DNA was preserved at −20 °C.Te concentration and quality of extracted genomic DNA were assessed using a spectrophotometer at the optimal densities of 280 nm and 260 nm [18].

Drug Susceptibility Testing. Following MTB genomic
DNA extraction using the PrimeXtract ™ kit instructions [25], the second-generation line probe assays (LPAs), MTBDRplus VER2.0 (to detect RIF and INH resistance), and MTBDRsl VER2.0 kit strips (to detect FLQs and aminoglycosides/peptide resistance) were performed following the manufacturer's protocol (Hain Lifescience GmBH, Nehren, Germany) [26,27].All INH and RIF-resistant TB isolates were tested using MTBDRsl VER 2.0 for detecting FLQs and second-line injectable drugs (SLIDs)-resistant isolates.For each run of LPAs, MTB strains H37Rv susceptible to all anti-TB drugs tested and molecular-grade water were used as a positive and negative control, respectively.

Prospective Follow-Up Study.
A prospective follow-up study on 438 PTB-symptomatic individuals with culturenegative test results while at HWSs was done to determine the prevalence of developing active TB disease subsequent to residing at HWS. Te duration of follow-up was 12 months, starting on the date that a negative culture result was obtained.TB status was confrmed via telephone or in-person contact, and any reports of active TB were confrmed by reviewing the patient's medical records at the diagnosing public healthcare facility.Te date of diagnosis, the diagnostic method (acid-fast bacilli (AFB) smear microscopy, GeneXpert ® MTB/RIF assay, LPAs, or other diagnoses, like chest X-rays, and clinical diagnoses), the diagnostic public healthcare facility, and the location (zone) were recorded from participants who reported developing active TB disease during the follow-up period.   2 and Table S2).

Risk Factors for Culture-Positive Pulmonary Tuberculosis.
Te bivariate logistic regression analysis revealed that participants aged 34-49 years ((crude odds ratio (cOR) 0.44; 95CI: 0.28-0.70)),and rural residents (cOR 1.55; 95CI: 1.03-2.34)were statistically associated with culture-positive PTB.Additionally, the analysis revealed that few independent variables were statistically associated with culture-positive PTB (p < 0.05), including a history of TB disease, contact with chronic coughers or active TB patients, having had close contact with a family member who has TB, the number of days (>21) spent at HWS, and sharing living spaces (rooms) at HWS (Table S3).
In the fnal multivariate logistic regression model, place of residence, marital status, family size per household, and sharing a living space (rooms) at HWSs were positively associated with culture-positive PTB.Rural residents were two times more likely to develop culture-positive PTB compared to urban residents (aOR 2.65; 95CI: 1.38-5.10).Married participants were more likely to have culturepositive PTB than single participants (aOR 2.43; 95CI: 1.28-4.63).On the other hand, participants with more than fve family members per household were 1.84 times more likely to have culture-positive PTB than those with less than fve (aOR 1.84; 95CI:1.04-3.24).Besides, sharing a living space at HWS increased the risk of developing culturepositive PTB by tenfold (aOR 10.57; 95CI: 3.60-31.13)(Table 3).

Risk Factors Associated with Drug
Resistance.Te multivariate logistic regression analysis revealed that a history of TB disease and study area has a signifcant association with the occurrence of some form of drug-resistant TB.Attendees who had a history of TB disease were nine times more likely to sufer from drug-resistant TB as compared with other HWS attendees who had no history of TB (aOR 9.22; 95 CI: 1.55-54.82).On the other hand, participants who attended HWS in the South Wello zone were three times more likely to develop any drug-resistant TB as compared with those who attended HWS in Central Gondar (aOR 3.06; 95 CI: 0.21-6.72).Similarly, the analysis revealed that attendees' occupations and study areas have a signifcant association with the occurrence of MDR-TB.Tus, farmers and housewives were nine (aOR 9.78; 95 CI: 1.55-61.59)and ffteen (aOR 15.68; 95 CI: 1.46-168.10)times more likely to develop MDR-TB as compared with students, respectively.Moreover, participants who attended HWS in the South Wello zone were twice as likely to develop MDR-TB as compared to those who attended HWS in Central Gondar (aOR 2.08; 95 CI: 0.31-3.92)(Table 5).
In addition, the proportion of any drug-resistant and MDR-TB in each study zone was estimated based on the participant's age category and found that participants aged 18-33 years appeared to be the most afected.Tus,  S4).On the other hand, the logistic regression analysis showed that the odds of developing any drug-resistant TB and MDR-TB were 14.54 (95 CI: 1.65-128.44)and 12.00 (95% CI: 1.35-106.80)times higher in the South Wello zone compared to the North Wello zone (Table S5).

Te Prospective Follow-Up Study Results.
A prospective follow-up study on 438 PTB-symptomatic individuals with culture-negative tests while at HWS was done to determine the proportion of developing active TB disease subsequent to residing at HWS. Te duration of follow-up was 12 months, starting on the date that a negative culture result was obtained.Among 438 participants who were on followup, 30 (6.8%) (95 CI 4.4-9.4%)developed active TB disease post-residency at the HWS.Male participants and those   S7).

Discussion
In Ethiopia, HWSs are traditional places of healing where people travel all over to seek the restorative benefts of holy water blessed by Orthodox priests [13,16,19].In the study region, people use faith-based therapy with spiritual holy water for diferent diseases [13,16,17].Although studies have shown that TB patients seek treatment from traditional healers and HWSs, the burden of TB among HWS attendees in Ethiopia has not been thoroughly studied.
In this study, the prevalence of culture-positive PTB was 21.8%.Tus, the point prevalence was 1,183 per 100,000 attendees, which was 4.3 times higher than a national TB prevalence study in Ethiopia, which reported 277 per 100,000 bacteriologically confrmed TB cases and 108 per 100,000 smear-positive TB cases [28].Similarly, our fnding was 1.5-fold higher than a previous study that reported 795 per 100,000 HWS attendees had TB in the study region [13].Te same study reported that the prevalence of smearpositive PTB among adult HWS attendees was 7.4-fold higher than Ethiopia's national TB prevalence [13].Tis International Journal of Microbiology discrepancy may be due to the study population and laboratory diagnosis methods.Te national TB prevalence survey report in Ethiopia excluded congregate settings and high-risk groups found in the HWSs [28].On the other hand, Derseh and his colleagues used sputum smear microscopy, which has low sensitivity for TB detection, resulting in a low-prevalence fnding report [13].Te high prevalence of culture-positive PTB at HWSs may be due to overcrowding, close contact, inadequate ventilation of shared living spaces, and long stays, which increase exposure and TB transmission [13].People who visit HWS choose to treat their diseases religiously, including TB and HIV/AIDS, and perceive spiritual HWS as their best treatment choice [13,16,18].In addition, people in rural areas and poor communities lack knowledge of TB and often misinterpret TB symptoms as signs of other diseases [14].Another possible explanation for the high prevalence of PTB in our study and the report of a previous study of a similar nature [13] could be due to study setting selection biases since persons with TB symptoms are more likely to visit HWS to treat the disease spiritually [18,29,30].Te prevalence of culture-positive PTB (21.8%) in the current study was also higher than that found in other high-risk settings in Ethiopia, including prisons, homeless shelters, and university students [31][32][33][34][35][36][37][38].Te diference may be due to the fact that these studies used sputum smear microscopy, whereas we used conventional culture methods, which are more sensitive and specifc for TB diagnosis.Another probable explanation is that everyone who attends HWSs is prone to TB and other infectious diseases due to their various health conditions.In addition, the availability of TB diagnosis and treatment services in prisons and universities may enable the early diagnosis and treatment of TB cases.Our study result was also higher than community-based studies in Southern Ethiopia's rural districts, which reported 3.0 to 6.3% [39][40][41]; Amhara region (3.8 to 4.9%) [11,12]; Oromia region (7.6%) [42]; Tigray, Northern Ethiopia (8.6%) [43]; and central Ethiopia, Addis Ababa (13.3%) [44].Te diference may be due to diferences in the study populations (subnational versus HWS attendees), TB diagnostic methods (sputum smear microscopy versus culture technique), the study period, and study setting selection biases.
Te current study found that 18-33 years of age participants had the highest rate of culture-positive PTB.Tis may be because 47.0% of study participants were 18-33 years of age.Another possible explanation is that young people are more likely to regularly attend HWS and are hence likely to be exposed to TB infection.Most HWSs are in remote places, making it difcult for older adults to attend regularly.A similar study found that 68.6% of spiritual HWS attendees were 15-45 years of age [13].Te burden of TB among young 8 International Journal of Microbiology individuals has major health consequences, as these individuals are economically active, and their travel for employment and high social interactions within the community exacerbate TB transmission in the general population [45].Tis study found that rural residents are more likely to have culture-positive TB than urban residents.Tis was consistent with other studies' reports [46,47].According to global statistics, urban areas have a higher TB burden; however, TB is also prevalent among rural inhabitants in countries where a large portion of the population resides in rural areas and has a low income [48].Due to inadequate public healthcare facilities, poor TB services, individuals' poor healthcare-seeking behavior, and a lack of knowledge and information about TB, early diagnosis and treatment are especially difcult in rural areas [40].On the other hand, due to travel costs, fear of stigma, and other sociocultural and socioeconomic factors, people with TB in rural areas are unable to access healthcare facilities for early diagnosis and treatment [14,15].However, further research is required to investigate this interaction.
Te majority of participants with culture-positive TB in the current study were married.Tis was consistent with a previous study [48].In contrast to our fndings, a few studies have revealed that unmarried people have a higher risk of TB infection than married people [46,49,50].Tis might be because single people have a diferent lifestyle than married individuals.However, further study is necessary to better understand the factors that infuence marital status as a predictor of active TB.
Household size (>5 family members) was statistically associated with culture-positive TB in the current study.Tis was consistence with a previous similar study [13] and institution-based studies in southeast Ethiopia [49,51].Tis may be because poverty, malnutrition, and overcrowded living conditions all increase the risk of TB transmission [5].Since TB is mainly transmitted indoors, having a large family size per household results in overcrowding and increases the risk of TB transmissions.Another possible explanation is that people who live in rural areas and have many family members are more likely to be of lower socioeconomic status.
In our study, participants who shared living space at HWSs were tenfold more likely to have culture-positive PTB.Tis is because rooms at the HWSs are built as temporary waiting spaces, and they are very small, overcrowded, and poorly ventilated, which can intensify the risk of TB transmission [13,18,52].Te overcrowded and poorly ventilated waiting rooms at the HWS, prolonged stays at the HWS, and poor healthcare-seeking behaviors of individuals worsen the active transmission of TB at the HWS.Our fndings were comparable to those of prior studies [32,34,35].Congregate settings, such as HWSs, are places where people live together, and it is common for people to share living space [13,18,52].Hence, poor living conditions and overcrowding in the shared room can increase TB transmission.
Moreover, our study found 12.3% MDR-TB (resistance to both RIF and INH), suggesting that MDR-TB is also a major concern in these high-risk groups.Furthermore, 7.1% (3/42 retreated cases) of RR/MDR-TB isolates were identifed in previously treated, while 15% (12/80) were isolated from newly diagnosed TB cases.Our result was higher than earlier studies in the study region (1.0 to 8.4%) [53,54,58,59,70,71] and elsewhere in Ethiopia (1.2% to 8.3%) [64, 67-69, 72, 73].It was also higher than 4.4% in the Ethiopian national survey [66], 0.71% in a recent national report on MDR-TB among new cases [1], and prison settings in Ethiopia (9.5%) [74].However, it was comparable to previous studies from central Ethiopia, Addis Ababa [61,75], Eastern Ethiopia [63], and a recent Ethiopian national report on MDR-TB among retreated TB patients (12.0%) [1].Te discrepancy may be due to diferences in study populations, study settings, and DST methods used to diagnose MDR-TB.Te high prevalence of DR-TB, particularly MDR-TB, among HWS attendees in the study region indicated that many TB patients, including those infected with DR-TB strains, attended the sites and that DR-TB strain transmission is common.However, molecular epidemiology studies are necessary to understand resistant TB strain transmission in HWS attendees and the community.
In this study, 4.1% of isolates were FLQs-resistant.Interestingly, all fve FLQs-resistant and/or pre-XDR-TB isolates were identifed at the South Wello zone.Tis suggests that these pre-XDR-TB strains may be circulating in this study site and have recently disseminated, although a molecular epidemiology analysis with strong discriminatory power would be required to confrm genotypic similarities, and recent transmission [18,52].Consistent with our result, an earlier study conducted in the same study region found that 5.7% of TB patients had pre-XDR-TB strains [7].Similarly, a multicenter study revealed that the prevalence of pre-XDR-TB in Ethiopia was 5.0% [76].However, both studies and ours had quite diferent study populations.Shibabaw and his colleagues included all RIF-resistant or MDR-TB patients who attended MDR-TB treatment centers before starting anti-TB therapy [7], while Dagne and his colleagues included new and retreated TB cases who attended TB treatment centers in diferent Ethiopian settings [76].Tus, our study demonstrated that DR-TB, particularly MDR-TB, and pre-XDR-TB, among PTB-symptomatic HWS attendees in Ethiopia is a major issue that International Journal of Microbiology necessitates urgent prevention intervention measures and more studies on the same high-risk groups.
In the present study, participants between the ages of 18 and 33 years appeared to be at the highest risk of any DR-TB and MDR-TB infection, with rates of 47.1% and 58.3%, respectively.Although the comparison is difcult since diferent studies use diferent age cutof points, an earlier study in the same study region found a strong link between anti-TB drug resistance and the age range of 25 to 34 years [58].Similarly, a study conducted in South Africa revealed a strong link between DR-TB and the age groups 35 to 54 years and over 55 years [77].Young age groups' increased exposure to the external environment, high-risk behavior, high workload, and broad range of mobility might contribute to TB and DR-TB infection.Te high rate of DR-TB among younger folks has major health repercussions, as these individuals are economically active, and their travel for employment and high social contacts within the community exacerbates TB transmission in the general population [45].
In addition, in the current study, the South Wello zone study area appears to have the highest rate of any DR-TB and MDR-TB, with the odds of 14.5-and 12.0-fold higher than other study sites, respectively.Interestingly, fve FLQsresistant and/or pre-XDR-TB strains were identifed at the South Wello zone study site.Tis could be because DR-TB is prominent in this study area and there is recent TB transmission, although further molecular epidemiology analysis with strong discriminatory power would be required to confrm genotypic similarities and transmission patterns of the resistant strains [18,52].
In the prospective follow-up study, a signifcant proportion of participants reported the development of active TB disease following exposure to HWS.Although our prospective follow-up method has limitations (unable to collect specimens for further laboratory confrmation), it suggests that HWS is a high-risk setting for TB transmission.Tus, these individuals can spread the disease to the community and their household members.We found that the attendee's educational status and sex, having had close contact with active TB patients in their family members, sharing living space at HWS, and recent hospitalization were potential risk factors for developing active TB disease postresidency at the HWS.TB transmission will be enhanced in environments where social mixing is more likely (along with overcrowding).Similarly, factors that prolong an infectious patient's exposure time will enhance TB transmission to other individuals [78].

Limitations.
Tere were a few limitations to our study.First, there is a population selection bias and likely information bias when using self-reported data on risk factors.Second, the confrmatory tests were not done on those who reported active TB progression post-residency at the HWSs.Besides, in the follow-up study cohort, a comparison group of people who never attended or had no HWS exposure is crucial to determine the level of risk of HWS exposure to acquiring active TB post-residency at the HWS.Tird, due to fnancial constraints, we were unable to perform BD BACTEC MGIT 960 for the initial MTB isolation process, and no phenotypic DST (pDST) was done on these isolates because liquid culture techniques, especially MGIT 960, have a signifcant advantage over solid culture techniques in terms of turnaround time and better recovery of MTB isolates.Last, further molecular epidemiology analysis would be warranted to confrm the transmission patterns of TB strains among HWS attendees.

Conclusions
Given that the prevalence of PTB among HWS attendees in this study population was seen to be higher than in the general population, hence, proactive preventive measures are recommended.Te current study revealed that rural residents, being married, having >5 family members, and sharing a living space at HWSs were predictors of culturepositive PTB.Te follow-up study also revealed that a higher proportion of attendees developed active TB disease postresidency at the HWS.Furthermore, the study showed a higher rate of DR-TB, especially MDR-TB, and pre-XDR-TB, among HWS attendees.Participants aged 18-33 years and the South Wello zone study site appeared to be more afected by DR-TB strains.Tus, regional and national TB prevention and control programs should recognize HWSs as high-risk settings for TB transmission and implement regular systematic TB screening, detection of DR-TB strains, and routine drug resistance surveillance among HWS attendees.Robust collaboration between the Ethiopian Orthodox Tewahedo Church and the regional and national TB control program is essential to develop locally appropriate, culturally accepted, and efective policy interventions to halt TB transmission among HWS attendees and the community.

Data Availability
Te data sets analyzed during this study are available from the corresponding author upon reasonable request.

Disclosure
A poster presentation was made at the Union World Conference on Lung Health 2022, and the corresponding abstract has been published and is available at https:// theunion.org/sites/default/fles/2022-11/Abstract_Book_2022-compressed.pdf[79], University of Pretoria, and Woldia University (only for scholarship and laboratory fees).Te funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.
International Journal of Microbiology

Table 3 :
Multivariate logistic regression analysis of potential associated risk factors for culture-positive TB among PTB-symptomatic HWS attendees (n � 560).

Table 4 :
Te proportion of drug-resistant TB isolates and characteristics of study participants (n � 122).

Table 5 :
Factors associated with any drug-resistant TB (RIF r and/or INH r ) and RIF r /MDR-TB.